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Dental caries is a microbial disease and the most common chronic health condition, affecting nearly 3.5 billion people worldwide. In this study, we used a multiomics approach to characterize the supragingival plaque microbiome of 91 Australian children, generating 658 bacterial and 189 viral metagenome-assembled genomes with transcriptional profiling and gene-expression network analysis. We developed a reproducible pipeline for clustering sample-specific genomes to integrate metagenomics and metatranscriptomics analyses regardless of biosample overlap. We introduce novel feature engineering and compositionally-aware ensemble network frameworks while demonstrating their utility for investigating regime shifts associated with caries dysbiosis. These methods can be applied when differential abundance modeling does not capture statistical enrichments or the results from such analysis are not adequate for providing deeper insight into disease. We identified which organisms and metabolic pathways were central in a coexpression network as well as how these networks were rewired between caries and caries-free phenotypes. Our findings provide evidence of a core bacterial microbiome that was transcriptionally active in the supragingival plaque of all participants regardless of phenotype, but also show highly diagnostic changes in the ways that organisms interact. Specifically, many organisms exhibit high connectedness with central carbon metabolism to Cardiobacterium and this shift serves a bridge between phenotypes. Our evidence supports the hypothesis that caries is a multifactorial ecological disease.

Antibiotic overuse has promoted the spread of antimicrobial resistance (AMR) with significant health and economic consequences. Surveillance of resistance reservoirs, like the rarely explored oral microbiome, is necessary to combat AMR. We characterised the development of the paediatric oral resistome and investigated its role in dental caries in 221 twin children sampled over the first decade of life. From 535 oral metagenomes, we identified 309 antimicrobial resistance genes (ARGs), which significantly clustered by age, with the influence of host genetics on antimicrobial resistance increasing over time. Our results suggest the mobilisation potential of ARGs increased with age as Tn916 transposase was co-located with more species and ARGs as children grew. Dental caries compared to health was associated with a depletion of ARGs and species, which was reversed when restored. Our results highlight the dynamic nature of the paediatric oral resistome, and its potential role in transmission of AMR and dysbiosis.

Humans are host to a multitude of microorganisms that rapidly populate the body at birth, subject to a complex interplay that is dependent on host genetics, lifestyle, and environment. The host-associated microbiome, including the oral microbiome, presents itself in a complex ecosystem important to health and disease. As the most common chronic disease globally, dental caries is induced by host-microbial dysbiosis in children and adults. Multiple biological and environmental factors are likely to impact disease predisposition, onset, progression, and severity, yet longitudinal studies able to capture these influences are missing. To investigate how host genetics and environment influenced the oral microbial communities over time, we profiled supragingival plaque microbiomes of dizygotic and monozygotic twins during 3 visits over 12-months. Dental plaque DNA samples were amplified by targeting the 16S rRNA gene V4 region, and microbial findings were correlated with clinical, diet and genetic metadata. We observed that the oral microbiome variances were shaped primarily by the environment when compared to host genetics. Among the environmental factors shaping microbial changes of our subjects, significant metadata included age of the subject, and the age by which subjects initiated brushing habits, and the types of actions post-brushing. Relevant heritability of the microbiome included Actinomyces and Capnocytophaga in monozygotic twins and Kingella in dizygotic twins. Corynebacterium and Veillonella abundances were associated with age, whereas Aggregatibacter was associated with younger subjects. Streptococcus abundance showed an inverse association over time, and Selenomonas abundances increased with brushing frequency per day. Unraveling the exact biological mechanisms in caries has the potential to reveal novel host-microbial biomarkers, pathways, and targets important to effective preventive measures, and early disease control in children.

Children’s oral health is in a dire state, with dental decay (caries) being one of the most common chronic diseases. While the role of bacteria in the oral microbiome and dental caries is established, the contribution of fungi is relatively unknown. We assessed the oral mycobiome in childhood (n = 17), to determine if the composition of fungi varies between children with and without caries. Oral mycobiome composition was assessed by using Illumina MiSeq to sequence the ITS2 region, which was amplified from dental plaque. This revealed that the oral mycobiome in the investigated children contained 46 fungal species. Candida albicans was the most abundant species and was ubiquitous in all samples, indicating this species may not be involved in caries development as previously suggested. While the overall diversity of fungi was similar, independent of caries status (p > 0.05), we found caries influenced the abundance of specific fungi. Children without caries had a significantly higher abundance of 17 species compared to children with caries, which had three enriched species (p < 0.001). While the differentially abundant species between health and caries may be specific to an Australian population, our findings indicate the mycobiome plays a role in oral health.

Next-Generation Sequencing is providing insights into the critical role of the oral microbiome in dental diseases. Application of this method can require the collection of dental plaque from large cohorts in field-type conditions, which necessitates a transport medium to preserve the microbiome composition. We evaluated the use of two transport media, VMG II and RNAprotect® Bacteria Reagent (Qiagen), for room temperature storage of dental plaque. VMG II has not previously been assessed for suitability to store microbiome samples intended for deep sequencing. We compared the microbiome composition of dental plaque (total n=23) stored in either VMG II or RNAprotect Bacteria at room temperature with immediately-frozen plaque. 454 sequencing of 16S gene amplicons was used to assess the plaque microbial composition. While the bacterial diversity recovered was similar between storage conditions (p>0.1), the abundance of bacteria was influenced by storage environment. Dental plaque stored in VMG II was most similar to immediately-frozen material, with only one of the 324 bacterial species being differentially abundant (Neisseria, p

Host-associated microbial communities are influenced by both host genetics and environmental factors. However, factors controlling the human oral microbiome and their impact on disease remain to be investigated. To determine the combined and relative effects of host genotype and environment on oral microbiome composition and caries phenotypes, we profiled the supragingival plaque microbiome of 485 dizygotic and monozygotic twins aged five to eleven. Oral microbiome similarity always increased with shared host genotype, regardless of caries state. Additionally, although most of the variation in the oral microbiome was determined by environmental factors, highly heritable oral taxa were identified. The most heritable oral bacteria were not associated with caries state, did not tend to co-occur with other taxa, and decreased in abundance with age and sugar consumption frequency. Thus, while the human oral microbiome composition is influenced by host genetic background, potentially cariogenic taxa are likely not controlled by genetic factors.

The Craniofacial Biology Research Group in the School of Dentistry at The University of Adelaide is entering an exciting new phase of its studies of dental development and oral health in twins and their families. Studies of the teeth and faces of Australian twins have been continuing for nearly 30 years, with three major cohorts of twins recruited over that time, and currently we are working with twins aged 2 years old to adults. Cross-sectional data and records relating to teeth and faces of twins are available for around 300 pairs of teenage twins, as well as longitudinal data for 300 pairs of twins examined at three different stages of development, once with primary teeth, once at the mixed dentition stage, and then again when the permanent teeth had emerged. The third cohort of twins comprises over 600 pairs of twins recruited at around birth, together with other family members. The emphasis in this third group of twins has been to record the timing of emergence of the primary teeth and also to sample saliva and dental plaque to establish the timing of colonization of decay-forming bacteria in the mouth. Analyses have confirmed that genetic factors strongly influence variation in timing of primary tooth emergence. The research team is now beginning to carry out clinical examinations of the twins to see whether those who become colonized earlier with decay-forming bacteria develop dental decay at an earlier age. By making comparisons within and between monozygotic (MZ) and dizygotic (DZ) twin pairs and applying modern molecular approaches, we are now teasing out how genetic, epigenetic, and environmental factors interact to influence dental development and also oral health.

Background: Information on the timing and sequence of human tooth emergence is valuable when analysing human growth and development, predicting the age of individuals, and for understanding the effects of genetic and environmental influences on growth processes. This paper provides updated data on the timing and sequence of primary tooth emergence in Australian children for both clinicians and researchers. Methods: Twins were recruited from around Australia with data collected through parental recording of twins’ primary tooth emergence. One twin from each pair was then randomly selected to enable the calculation of descriptive statistics for timing, sequence and asymmetry in tooth emergence. Results: The first and last primary teeth emerged, on average, at 8.6 months and 27.9 months, respectively, with teeth emerging in the order: central incisor, lateral incisor, first molar, canine, second molar. Left-side antimeric teeth were more likely to emerge before their right-side counterparts but this was not statistically significant. At least 35% of all antimeric pairs had emerged within two weeks of each other, serving as a useful guideline for assessing symmetrical versus asymmetrical development. Conclusions: Primary tooth emergence in Australian twins is occurring later than reported previously for Australian singletons but is consistent with findings for singletons in other ethnic groups. The most common sequence of primary tooth emergence appears to be consistent in twins and singletons and has not changed over time.

Utilising data derived from twins and their families, different approaches can be applied to study genetic and environmental influences on human dental variation. The different methods have advantages and limitations and special features of the twinning process are important to consider. Model-fitting approaches have shown that different combinations of additive genetic variance (A), non-additive genetic variance (D), common environmental variance (C), and unique environmental variance (E) contribute to phenotypic variation within the dentition, reflecting different ontogenetic and phylogenetic influences. Epigenetic factors are also proposed as important in explaining differences in the dentitions of monozygotic co-twins. Heritability estimates are high for most tooth size variables, for Carabelli trait and for dental arch dimensions, moderate for intercuspal distances, and low for some occlusal traits. In addition to estimating the contributions of unmeasured genetic and environmental influences to phenotypic variation, structural equation models can also be used to test the effects of measured genetic and environmental factors. Whole-genome linkage analysis, association analysis of putative candidate genes, and whole genome association approaches, now offer exciting opportunities to locate key genes involved in human dental development.

Our understanding of tooth eruption in humans remains incomplete. We hypothesized that genetic factors contribute significantly to phenotypic variation in the emergence of primary incisors. We applied model-fitting to data from Australian twins to quantify contributions of genetic and environmental factors to variation in timing of the emergence of human primary incisors. There were no significant differences in incisor emergence times between zygosity groups or sexes. Emergence times of maxillary central incisors and mandibular lateral incisors were less variable than those of maxillary lateral incisors and mandibular central incisors. Maxillary lateral incisors displayed significant directional asymmetry, the left side emerging earlier than the right. Variation in timing of the emergence of the primary incisors was under strong genetic control, with a small but significant contribution from the external environment. Estimates of narrow-sense heritability ranged from 82 to 94% in males and 71 to 96% in females.

Our studies of the teeth and faces of Australian twins commenced at the School of Dentistry, The University of Adelaide in the early 1980s. There are now over 900 pairs of twins enrolled in our continuing investigations, together with 1200 relatives. There are 3 main cohorts of participants. The first cohort comprises around 300 pairs of teenage twins for whom various records have been collected, including dental casts, facial photographs, finger and palm prints and information on laterality, including handedness. The second cohort comprises around 300 pairs of twins who have been examined at 3 stages of dental development from approximately 4 years of age to about 14 years: at primary, mixed, and permanent dentition (excluding 3rd molars) stages. The most recent study of tooth emergence and oral health, for which we are currently recruiting twins, will provide a third cohort of around 500 twin pairs aged from around birth to 3 to 4 years of age. Our broad aim in these studies has been to improve our understanding of how genetic and environmental factors contribute to variation in dental and facial features, and to oral health. We have also used our data to investigate aspects of the determination of laterality, particularly the fascinating phenomenon of mirror imaging. We plan to maximize the use of the longitudinal data and DNA we have collected, and continue to collect, by performing genome-wide scans for putative genetic linkage peaks for a range of dental features, and then to test for association between a series of likely candidate genes and our phenotypes.

Chorion type may significantly influence the prenatal environment of twins. This study explored the associations between chorion type and gestational age, birth weight, birth length, and the timing of emergence of the first primary tooth in two populations of twins, Australian and Dutch. Additionally, we investigated the relationship between chorion type and birth weight discordance (BWD) in order to determine whether a significant relationship existed between discordance in birth weight and discordance in the timing of emergence of the first primary tooth. The two study samples consisted of 409 Australian twin pairs and 301 Dutch twin pairs, all of European ancestry. Data were collected through a combination of questionnaires and recording charts administered to the parents and through linkage with biological databases. In the Australian sample, monozygotic monochorionic (MZMC) twins experienced the shortest mean gestation time (35 weeks), the lowest mean birth length (46 cm) and the lowest mean birth weight (2.3 kg) compared with other twin groups. For the same variables in the Dutch sample, these trends with MZMC twinning were not observed. Chorion type did not significantly affect the mean timing of emergence of the first primary tooth in either sample. Monochorionicity was found to be significantly associated with BWD in both samples, but there was a significant association between BWD in MZMC twin pairs and timing of emergence of the first primary tooth only in the Australian sample. Results from this study support previous findings that the timing of emergence of the first primary tooth is influenced strongly by genetic factors and is well protected from environmental disturbances.

The continuing studies of the teeth and faces of Australian twins and their families in the Craniofacial Biology Research Group in the School of Dentistry at the University of Adelaide began 30 years ago. Three main cohorts of twins have been recruited, enabling various objectives and specific hypotheses to be addressed about the roles of genetic, epigenetic and environmental influences on human dentofacial growth and development, as well as oral health. This paper highlights some key findings arising from these studies, emphasizing those of direct relevance to practising oral health professionals. We also draw on published literature to review the significant developments in relation to the use of precision 2D and 3D imaging equipment, the application of modern molecular techniques, and the development of sophisticated computer software for analysing genetic relationships and comparing complex shapes. Such developments are valuable for current and future work. Apart from the classical or traditional twin model, there are several other twin models that can be used in research to clarify the relative contributions of genetic, epigenetic and environmental contributions to phenotypic variation. The monozygotic (MZ) co-twin model is one particularly valuable method, given that examination of only one pair of MZ twins can provide considerable insights into underlying causes of observed variation. This model can be used in a dental practice environment, with oral health professionals having the opportunity to explore differences in orofacial structures between MZ co-twins who are attending as patients. As researchers have become more aware of the complexities of the interactions between the genome, the epigenome and the environment during development, there is the need to collect more phenotypic data and define new phenotypes that will better characterize variations in growth processes and health status. When coupled with powerful new genetic approaches, including genome-wide association studies and linkage analyses, exciting opportunities are opening up to unravel the causes of problems in craniofacial growth and common oral diseases in human populations.

Grant Townsend, Michelle Bockmann, Toby Hughes, Suzanna Mihailidis, W. Kim Seow and Alan Brook

The aim of this review is to highlight some key recent developments in studies of tooth number, size and shape that are providing better insights into the roles of genetic, environmental and epigenetic factors in the process of dental development. Advances in molecular genetics are helping to clarify how epigenetic factors influence the spatial and temporal regulation of the complex processes involved in odontogenesis. At the phenotypic level, the development of sophisticated systems for image analysis is enabling new dental phenotypes to be defined. The 2D and 3D data that are generated by these imaging systems can then be analysed with mathematical approaches, such as geometric morphometric analysis. By gathering phenotypic data and DNA from twins, it is now possible to use ‘genome-wide’ association studies and the monozygotic co-twin design to identify important genes in odontogenesis and also to clarify how epigenetic and environmental factors can affect this process. Given that many of the common dental anomalies affecting the human dentition are interrelated, apparently reflecting pleiotropic genetic effects, the discoveries and new directions described in this paper should have important implications for clinical dental practice in the future.

Findings are presented from a prospective cohort study of timing of primary tooth emergence and timing of oral colonization ofStreptococcus mutans(S. mutans) in Australian twins. The paper focuses on differences in colonization timing in genetically identical monozygotic (MZ) twins. Timing of tooth emergence was based on parental report. Colonization timing ofS. mutanswere established by plating samples of plaque and saliva on selective media at 3 monthly intervals and assessing colony morphology. In 25% of individuals colonization occurred prior to emergence of the first tooth. A significant proportion of MZ pairs (21%) was discordant for colonization occurring before or after first tooth emergence, suggesting a role of environmental or epigenetic factors in timing of tooth emergence, colonization byS. mutans, or both. These findings and further application of the MZ co-twin model should assist in development of strategies to prevent or delay infection withS. mutansin children.

The aim of this study was to quantify contributions of genetic and environmental factors to variation in timing of emergence of the primary teeth in a sample of monozygotic and dizygotic twins, using univariate model-fitting approaches. The sample comprised 94 pairs of monozygotic twins and 125 pairs of dizygous twins, all of European ancestry, aged from 2–6 years. Tooth emergence timing was based on parental report, with a subset of data validated by clinical assessment. Heritability estimates for tooth emergence timing were generally high, around 90%, however estimates for the lower right lateral incisor and the lower canines were around 50%. These findings confirm a strong genetic influence on observed variation in the timing of emergence of the human primary teeth.

Two metaphors are presented to highlight concepts that could lead to a paradigm shift in dental studies of twins. The first, derived from the Song of Solomon in the Bible, refers to teeth as being twins. This viewpoint emphasises that each tooth should be viewed as a paired structure, not only with its antimere (within the same arch) but also with its isomer (in the opposing arch). The other metaphor provided by Waddington in 1957 is visual and involves 'an epigenetic landscape' that represents the processes of decision-making by cells during development. It likens the different stages of cellular decision-making to a ball rolling down an undulating landscape of interconnecting hills and valleys. This viewpoint helps to explain how distinct differences in dental phenotypes may arise both within and between monozygotic (MZ) co-twins due to relatively minor temporospatial effects during development. Measurements of maximum mesiodistal diameters of teeth in a pair of MZ twins, using calipers and also 2D and 3D imaging systems, have demonstrated that differences in dental crown size occur between antimeric pairs and between corresponding teeth of MZ co-twins. By defining new dental phenotypes that provide more comprehensive descriptions of tooth size and shape, and by drawing on the metaphors described, we are confident of providing new insights into the reasons for observed similarities and differences within, and between, the dentitions of twins. Our approaches will focus on multivariate analyses that take into account the paired arrangement of teeth and also explore epigenetic, as well as genetic and environmental, sources of variation.

Two metaphors are presented to highlight concepts that could lead to a paradigm shift in dental studies of twins. The first, derived from the Song of Solomon in the Bible, refers to teeth as being twins. This viewpoint emphasises that each tooth should be viewed as a paired structure, not only with its antimere (within the same arch) but also with its isomer (in the opposing arch). The other metaphor provided by Waddington in 1957 is visual and involves ‘an epigenetic landscape’ that represents the processes of decision-making by cells during development. It likens the different stages of cellular decision-making to a ball rolling down an undulating landscape of interconnecting hills and valleys. This viewpoint helps to explain how distinct differences in dental phenotypes may arise both within and between monozygotic (MZ) co-twins due to relatively minor temporospatial effects during development. Measurements of maximum mesiodistal diameters of teeth in a pair of MZ twins, using calipers and also 2D and 3D imaging systems, have demonstrated that differences in dental crown size occur between antimeric pairs and between corresponding teeth of MZ co-twins. By defining new dental phenotypes that provide more comprehensive descriptions of tooth size and shape, and by drawing on the metaphors described, we are confident of providing new insights into the reasons for observed similarities and differences within, and between, the dentitions of twins. Our approaches will focus on multivariate analyses that take into account the paired arrangement of teeth and also explore epigenetic, as well as genetic and environmental, sources of variation.

Aims: This study is part of a larger investigation of genetic and environmental influences on primary tooth emergence in Australian twins. Our aims were to describe patterns of emer

Background: Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disorder related to a deficiency in the enzyme iduronate-2-sulfatase (IDS). Clinical trials of enzyme replacement therapy are in progress, but effective treatment will require screening assays to enable early detection and diagnosis of MPS II. Our study evaluated the diagnostic accuracy of IDS protein and enzyme activity measurements in dried blood spots and plasma. Methods: We collected dried-blood-spot and plasma samples from unaffected control individuals and from MPS II patients. We measured IDS protein concentration with a 2-step time-delayed dissociation-enhanced lanthanide fluorescence immunoassay. To measure enzyme activity, we immobilized anti-IDS antibody on microtiter plates to capture the enzyme and measured its activity with the fluorogenic substrate 4-methylumbelliferyl sulfate. Results: Dried-blood-spot samples from MPS II patients showed an almost total absence of IDS activity (0–0.075 μmol · h−1 · L−1) compared with control blood spots (0.5–4.7 μmol · h−1 · L−1) and control plasma (0.17–8.1 μmol · h−1 · L−1). A dried-blood-spot sample from only 1 of 12 MPS II patients had detectable concentrations of IDS protein (24.8 μg/L), but no IDS protein was detected in plasma from MPS II patients. Ranges for IDS protein in control samples were 25.8–153 μg/L for blood spots and 22.8–349.4 μg/L for plasma. Conclusion: Measurement of the IDS protein concentration and enzyme activity (as measured by a simple fluorogenic assay with an immune capture technique) enables identification of the majority of MPS II patient samples from both dried blood spots and plasma samples.

Lysosomal storage disorders (LSD) are chronic progressive diseases that have a devastating impact on the patient and family. Most patients are clinically normal at birth but develop symptoms early in childhood. Despite no curative treatment, a number of therapeutic options are available to improve quality of life. To achieve this, there is a pressing need for newborn screening to identify affected individuals early, before the onset of severe irreversible pathology. We have developed a multiplexed immune-quantification assay of 11 different lysosomal proteins for the identification of individuals with an LSD and evaluated this assay in a retrospective study using blood-spots from; newborns subsequently diagnosed with an LSD (n = 19, six different LSD), individuals sampled after diagnosis of an LSD (n = 92, 11 different LSD), newborn controls (n = 433), and adult controls (n = 200). All patients with mucopolysaccharidosis type I (MPS I), MPS II, MPS IIIA, MPS VI, metachromatic leukodystrophy, Niemann-Pick disease type A/B, and multiple sulfatase deficiency could be identified by reduced enzyme levels compared to controls. All mucolipidosis type II/III patients were identified by the elevation of several lysosomal enzymes, above the control range. Most Fabry, Pompe, and Gaucher disease patients were identified from either single protein differences or profiles of multiple protein markers. Newborn screening for multiple LSD is achievable using multiplexed immune-quantification of a panel of lysosomal proteins. With further validation, this method could be readily incorporated into existing screening laboratories and will have a substantial impact on patient management and counseling of families.

Background Mucopolysaccharidosis type VI (MPS VI) is a lysosomal storage disorder (LSD), which is caused by a deficiency in the enzyme N-acetylgalactosamine 4-sulfatase (4-sulfatase). MPS VI is characterized by severe skeletal abnormalities, somatic tissue pathology and early death. Treatment possibilities include bone marrow transplantation (BMT) and enzyme replacement therapy (ERT; currently in phase III clinical trial). Early diagnosis of MPS VI will allow treatment before the onset of irreversible pathology. Methods Sensitive immune assays have been developed to detect 4-sulfatase protein and activity in normal control and MPS VI blood-spots. Results Dried blood-spots from MPS VI patients contained no detectable 4-sulfatase protein and activity, compared to 3.5–21 μg/L of 4-sulfatase protein and 291–1298 nmol/min/L of activity for normal human controls. In this evaluation study, the assay was sensitive and 100% specific, allowing reliable detection of individuals with MPS VI. Conclusions The assays reported here have the potential to detect MPS VI patients using dried blood-spots.

Barrett and Brown studied tooth emergence in Aborigines living at Yuendumu in the Northern Territory of Australia (Barrett, 1957. Aust. Dent. J. 2, 217–227; Barrett, et al., 1964. Am. J. Phys. Anthropol. 22, 79–89; Brown, 1978. Ann. Hum. Biol. 5, 41–54). They showed that emergence timing of anterior primary teeth was delayed during the first year after birth but there was an increase in tempo in the second year. Emergence of anterior permanent teeth was also delayed in Aborigines but emergence of posterior teeth occurred earlier, third molars appearing at around 16 years of age. Although the sequence of emergence showed considerable variability, it was similar to other ethnic groups. Subsequently, we have investigated tooth emergence in singletons and twins of European ancestry. Analysis of records of over 8,500 Australian children showed that emergence of permanent teeth occurred later than reported previously (Diamanti and Townsend, 2003. Aust. Dent. J. 48, 39–42). We are now exploring primary tooth emergence in Australian twins, based on parental reports, and data for 190 pairs of twins have been analysed. The first teeth to emerge are generally the mandibular central incisors at around 8.7 months after birth, while the maxillary second molars are usually last at around 27.2 months. The most common sequence of emergence is central incisors, lateral incisors, first molars, canines and then second molars. Differences in emergence timing between the Aboriginal and non-Aboriginal samples reflect genetic and environmental effects, with infant mortality and morbidity and local factors within the dentition being important.